Validation of rapid fecal calprotectin assay using particle enhanced turbidimetric immunoassay for inflammatory bowel disease

Fecal calprotectin (FC) is a promising biomarker for diagnosis and treatment of inflammatory bowel disease, ulcerative colitis (UC), and Crohn’s disease. An enzyme immunoassay (EIA) is widely used for FC detection, though the considerable lag time, up to several days, causes clinical management delay. This study was performed to examine the new rapid kit fCAL-turbo, which is based on a particle-enhanced turbidimetric immunoassay (15 min), by comparing FC values with other EIAs (EliA, PhiCal, Bühlmann) and endoscopic scores. Using 94 samples, fCAL-turbo showed strong significant positive correlations with the other kits (Spearman’s r = 0.9178–0.9886). Of 74 UC patients, 69 underwent an endoscopy and fCAL-turbo reflected endoscopic activity with a moderate correlation with Mayo endoscopic subscore (MES) (r = 0.6945, others r = 0.6682–0.7013). Receiver operating characteristic analyses based on MES 0 versus 1–3 showed a similar efficacy as compared to the other kits (cut-off and area under the curve: 89.70 µg/g and 0.8592, respectively, others 62.35–138.4 µg/g and 0.8280–0.8611, respectively). Furthermore, multiple regression analysis confirmed that fCAL-turbo results significantly contributed to prediction of MES 0 with a higher t-value as compared to the other biomarkers. fCAL-turbo showed strong correlations with the other kits and also demonstrated excellent performance for predicting endoscopic remission of UC.

We and others have also confirmed that FC is correlated with endoscopic remission in a manner superior to other blood biomarkers, such as C-reactive protein (CRP), hemoglobin, platelets, and white blood cell count (WBC) [16][17][18][19]21,27 . Moreoer, it has been reported that its level shows elevation at two to three months before clinical relapse in IBD patients 28 .Thus, FC is regarded as one of the best biomarkers for prediction of endoscopic remission and clinical relapse in patients with IBD.
Several kits for detection of FC are available.Because of its accuracy and accumulated evidence, enzyme immunoassay (EIA) testing procedures, such as enzyme-linked immunosorbent assay (ELISA), and fluoroenzyme and chemiluminescence immunoassays, are considered to represent the gold standard 11 .EIA testing uses batch analysis and is normally performed by a specialist outside of the treating hospital 29 , thus results can be delayed for up to several days despite the 2.5-h EIA run-time.The present study was conducted to validate fCAL-turbo, a newly introduced rapid automated FC detection kit, in an IBD patient population.An fCAL-turbo assay is conducted as a particle-enhanced turbidimetric immunoassay and can be performed by a general chemical analyzer typically employed by a hospital, with the result available in approximately 15 min 30 .However, its clinical utility for IBD cases has not been fully elucidated.Therefore, we aimed to validate the performance of the fCAL-turbo assay by comparing results with other standard EIA assays in IBD patients.Furthermore, its efficacy for correlations with endoscopic activity [31][32][33] and prediction of endoscopic remission in those patients was investigated.

Patient demographics
A total of 94 IBD patients (74 UC, 20 CD) were included in this study.Patient profiles are shown in Table 1.Wide range of patients in age (range 20-83 years old), disease activity (78.2% and 75.0% of remission, and 21.8% Table 1.Clinical and demographic characteristics of IBD patients in this study.IBD inflammatory bowel disease, UC ulcerative colitis, CD Crohn's disease, IQR interquartile range, GI gastrointestinal.a Assessment of clinical disease activity by using partial Mayo score for UC (remission: 0-2 points, active: 3-9 points) and Crohn's Disease Activity Index (CDAI, remission: 0-150 points, active: > 150 points).b Assessment of disease extent, disease location, and disease behaviour by using the Montreal classification.TNF tumor necrosis factor, MES Mayo endoscopic subscore, mSES-CD modified simple endoscopic score for CD.www.nature.com/scientificreports/and 25.0% of active in UC and CD, respectively) and medications (i.e.immunomodulators, steroid, biological drug) were included, allowing more universal validation of FC assays in variety of patients' conditions.All fecal samples (n = 94) were included in the first comparison analysis (FC values comparison among four kits) (Fig. 1).
For the second comparison (FC values vs. clinical and endoscopic activities), results of 22 cases were excluded, as one had multiple inflammatory polyps found by colonoscopy, which can elevate the fecal calprotectin level 34 , and one had collagen disease (polyarteritis nodosa), which can elevate the FC level, which left the remaining sample size of 20 CD patients too small for this comparison analysis.For comparison with endoscopic activities, results from three patients unable to undergo an endoscopy colonoscopy examination for personal reasons were excluded.Finally, a total of 69 UC patients were analyzed using Mayo endoscopic subscore (MES) (Fig. 1).

fCAL-turbo kit shows strong positive correlations with other kits
All four of the examined kits detected calprotectin in each of the fecal samples (n = 94).In the first comparison analysis using all samples, the median FC value varied significantly among the kits [median values: EliA 84.2a).As for average fold changes, fCAL-turbo showed a 2.0-, 1.2-, and 0.7-fold change of median FC value from EliA, Buhlmann-E, and PhiCal, respectively.Such variations among the kits were consistently observed for all disease conditions (MES 0-3) (Fig. 2b).Interestingly, the fCAL-turbo FC values tended to be lower with lower disease activities (MES 0-1) and higher with increased disease activities (MES 2-3), which suggests a characteristic advantage for use of fCAL-turbo to more specifically detect disease activities.Spearman's rank correlation analysis demonstrated that fCAL-turbo had a significantly positive correlation with each of the other kits [compared to: EliA (r = 0.9178, 95% CI 0.8775-0.9453),PhiCal (r = 0.9317, 95% CI 0.8978-0.9546),Buhlmann-E (r = 0.9886, 95% CI 0.9827-0.9925)](Fig. 2c, Table 2).
As expected, FC values for endoscopic remission were significantly lower as compared to those for clinical remission (data not shown).Receiver operating characteristic (ROC) curve analysis for differentiation between endoscopic remission (MES = 0) and endoscopic activity (MES = 1-3) showed that the efficacy of the fCALturbo kit was similar to that of the other standard kits for the present UC cases (Fig. 3c, Table 5).The optimal  The AUC value obtained with the fCAL-turbo kit for predicting endoscopic remission was compared with values obtained with Elia, PhiCal, and Buhlmann-E, and found to be not significantly different (p = 0.251, p = 0.355, and p = 0.780, respectively).These results indicate that fCALturbo is a promising FC kit for predicting remission with an efficacy similar to the other FC kits tested.Finally, other clinical biomarkers for IBD, such as CRP, erythrocyte sedimentation rate (ESR), hemoglobin, and others, also useful for predicting remission, were examined, which showed that the AUC obtained with each of each of those was significantly smaller than the fCAL-turbo result (Fig. 3d).Moreover, multiple regression analysis confirmed that fCAL-turbo was the most effective, with a significantly higher t-value for the biomarkers CRP, ESR, hemoglobin, WBC, platelets, and albumin (Table 6).www.nature.com/scientificreports/ to an acceptable range has been proposed as a formal intermediate treatment target 35 .Because of its significant advantage of immediate turnaround time, as well as compatibility and cost performance, use of the fCAL-turbo can enhance the clinical efficacy of treat-to target strategies employed in IBD practice.To date, three excellent studies have presented comparisons of values obtained with various kits including fCAL-turbo, though did not include endoscopic disease activity 29,30,36 .Oyaert et al. 36 compared the diagnostic accuracy of six different assays using fecal samples from patients with CD (n = 15), UC (n = 12), gastrointestinal diseases used as a control (n = 52), and rheumatologic disease (n = 26).All six assays including fCAL-turbo demonstrated excellent diagnostic accuracy with similar AUCs.In the study by Noebauer et al. 29 , fecal samples from 95 symptomatic children suffering from chronic diarrhea, abdominal pain, and bloody stool were analyzed using fCAL-turbo, Buhlmann-E, and Quantum Blue, and a good correlation between fCAL-turbo and the other assays was found.Furthermore, Nilsen et al. 30 compared FC values obtained with the fCAL-turbo and Buhlmann-E kits, and also analyzed variations between those two clinical chemistry analyzers, with a good correlation demonstrated.The present findings support those presented in these previous studies.In addition, we compared FC values and endoscopic disease activity to explore whether the fCAL-turbo assay can provide reliable detection at any disease stage.The findings showed detection of calprotectin at all stages of disease and clearly revealed endoscopic activity in IBD patients.
To the best of our knowledge, this is the first study to use endoscopic score for validation of performance of the fCAL-turbo kit.In UC patients, findings obtained with fCAL-turbo had a strong positive correlation with endoscopic activity (MES).The AUC value for the fCAL-turbo (0.85) for detecting endoscopic remission (MES = 0) was consistent with those of the other assays (0.77-0.86) examined in this study and presented in previous reports, such as PhiCal (cut-off: 180 µg/g; AUC 0.67) 24 , Buhlmann-E (cut-off: 201 µg/g, AUC 0.88) and Quantum Blue (cut-off: 150.5 µg/g; AUC 0.88) 26 , and EliA Calprotectin 2 (cut-off: 146.0 µg/g; AUC 0.777) 25 .Therefore, it is anticipated that the fCAL-turbo kit will be considered to be a reliable tool to monitor UC activity.While data for CD patients are not presented because of the small sample size (n = 20), promising results have shown that fCAL-turbo and other kits, except for EliA, reflect endoscopic activity well, as shown by modified simple endoscopic score for CD (mSES-CD), though the strength of the correlation was found to be moderate, weaker than that in UC patients, which is similar to results presented in previous reports 37,38 .Nevertheless, FC remains a promising biomarker for CD, as we and others have demonstrated its greater accuracy for predicting endoscopic remission in CD patients as compared to serum biomarkers 17,35 .Further comprehensive investigations are required 35 .
As noted in other reports 39,40 , the present findings showed kit-dependent variations, with a maximum 3.8fold difference previously reported 36,41 .Several factors can influence the FC value, such as age 42,43 , obesity and diet 43 , physical inactivity 44 , and mucus or blood in the fecal sample 45 , though calprotectin itself remains stable for up to one week at room temperature 22,[46][47][48] .Since the same fecal samples were examined with all of the assay kits in this study, those factors do not require consideration.Possible reasons for the variations noted include (1) different assay (method) principles, (2) different antibody for capture or detection, and/or (3) potentially different fecal supernatant extraction efficacy 35,39 .The Buhlmann-E and fCAL-turbo kits use the same antibody, and the strongest correlation was noted between them.Thus, even with different methods and extraction kits, the antibody may be an important factor for result consistency, as the various antibodies used in the different assays would be directed against different complexes of the FC protein.
A variety of cut-off levels for endoscopic remission detection have been reported 35 , which was also observed in the present study.FC cut-off values in UC patients range from 58 to 490 µg/g and in CD patients from 71 to 918 µg/g, though consensus has yet to be established 35 .The present findings suggest that the cut-off is correlated with the kit-dependent actual value for calprotectin, with the cut-off highest for PhiCal and followed in order by fCAL-turbo, Buhlmann-E, and EliA (Table 5), while the median level of actual calprotectin value was also highest for PhiCal, followed by fCAL-turbo, Buhlmann-E, and EliA (Fig. 2).To determine a more reliable cut-off level, investigators will need to consider kit-dependent variations as well.Clinicians are advised to use the same kit for monitoring of IBD activity in individual patients.
The present study has some limitations.First, other assays presently available were not included in the analyses, such as immuno-chromatographic 49,50 , colloidal gold aggregation 51 , and home-based 52,53 assays, because of their lower frequency of use and level of accuracy.Furthermore, endoscopic score for IBD activity assessment was used, as that is the current gold standard for monitoring endoscopic remission in IBD patients [31][32][33] , though it would be better to add histological assessment.These limitations should be taken into account when interpreting the results regarding the clinical efficacy of FC for IBD endoscopic scores.
In conclusion, fCAL-turbo, a new rapid fully automated FC kit based on a particle-enhanced turbidimetric immunoassay method, showed strong and significant correlations with other established standard FC kits.Furthermore, moderate correlation with endoscopic activity in UC patients was noted, equivalent to that seen with the other kits.In particular, fCAL-turbo showed excellent performance for predicting endoscopic remission in UC patients, the same as the other standard assays.Based on its advantages including rapid results (15 vs. 150 min) and cost performance (random access testing vs. batch analysis) (Table S1) 29 , use of the fCAL-turbo kit can significantly augment the clinical efficacy of a treat-to-target strategy for IBD cases.

Population
Patient enrollment was performed from October 2016 to November 2018 at Shimane University Hospital and Matsue Seikyo General Hospital in Japan as part of our previous prospective studies 17,18 .For the present investigation, fecal samples obtained in the previous studies were used.The maximum frozen storage period was three years and there were no more than two freeze-thaw cycles.Although calprotectin protein is known to www.nature.com/scientificreports/remain stable for several days even at room temperature and after freeze-thaw cycles 47,48 , the quality of the fecal samples was verified, and there were no significant differences for representative FC values between those previous studies and the present.Information for the enrolled patients was analyzed in a retrospective manner.Enrolled patients, as in our previous studies 17,18 , were individuals with a previously established diagnosis of UC or CD, and scheduled to undergo a colonoscopy or balloon-assisted endoscopy (BAE).Excluded were those with colon cancer, acute or chronic gastrointestinal infection including Clostridioides difficile and cytomegalovirus, an artificial anus, regular intake of aspirin and/or other nonsteroidal anti-inflammatory drugs that can induce mucosal injury and might increase FC levels 54 , or unable to provide a fecal sample.As for patient demographics, gender, age, disease activity, duration, location, and behavior, and concomitant medications being taken at the time of fecal collection were noted.Clinical UC or CD disease activity was evaluated on the day of the endoscopy examination by use of a partial Mayo score (pMayo, endoscopic subscore removed, remission: 0-2 points, active stage: 3-9 points) 55 or CD activity index (CDAI; remission: 0-150 points, active stage: > 150 points) 56 , respectively.Assessment of disease extent, location, and behavior was done using the Montreal classification 57 .
The study protocol was approved by the institutional review board of both hospitals (Shimane University review board/Hospital review board) and performed in adherence to the Helsinki Declaration.Each patient provided written informed consent for participation.

Fecal samples
Fecal samples were collected at the first bowel movement in the morning 22 within three days before a bowel preparation for a colonoscopy procedure, and brought to the hospital or sent by postal mail immediately after collection.Upon arrival, fecal samples were immediately frozen at − 20 °C without a preservative.The maximum time lag from fecal collection to frozen state was within 24 h, during which period calprotectin proteins in fecal samples have been proven to remain stable at room temperature 22,[46][47][48] .

Measurement of fecal calprotectin level
FC levels were determined with the same fecal sample using each of the following four kits, according to the protocol of manufacturer.Those included three standard kits; EliA ® Calprotectin 2 (Thermo Fisher Scientific, Sweden), PhiCal ® Calprotectin ELISA (Immundiagnostik, Germany) 49 , and Buhlmann-E (BÜHLMANN fCAL ® ELISA, Bühlmann, Switzerland), and also the new BÜHLMANN fCAL ® turbo kit (Bühlmann, Switzerland) 30 .All samples were measured in duplicate under the same pre-analytical conditions, with the mathematical mean used to reflect any imprecision for the patient samples.The characteristics of each kit are shown in Table S1.PhiCal assays were performed by SRL Inc. (Tokyo, Japan) as part of our previous prospective study, while the others included in the present study were performed by Nissui Pharmaceutical Co., Ltd.(Tokyo, Japan), after receiving the preserved fecal samples.

Endoscopic activity scoring
The UC and CD patients received a polyethylene glycol-or magnesium citrate-based electrolyte solution for bowel preparation prior to the endoscopy.In those with UC, a total colonoscopy was generally performed with a magnifying colonoscope (PCF 260AZI, Olympus, Tokyo, Japan).The findings were evaluated using MES for each of five portions of the colorectum (cecum to ascending colon, transverse, descending, sigmoid colon, rectum).Maximum MES in the colorectum was used as the final endoscopy score for the present study.In all patients with CD, ileocolonoscopy or retrograde BAE using a double-balloon enteroscope EN-450T5 (Fujifilm, Tokyo, Japan) examinations were performed.Endoscopic findings indicating CD were evaluated based on mSES-CD, in which the narrowing score is removed for CD cases.All of the endoscopic procedures were performed by experienced gastroenterologists, with the endoscopic scores independently re-assessed by two expert colonoscopists (K.K., A.O.) who were blinded to the FC results.If there were any discrepancies, the final accepted score was determined based on discussion with the two experts and their supervisor.

Statistical analysis
Nonparametric data are presented as median and interquartile range or 95% CI.For non-paired non-parametric comparisons, a Mann-Whitney test was performed (two-tailed).For non-paired non-parametric multiple comparisons, a Kruskal-Wallis test with ANOVA was performed (two-tailed), while correlation analyses were performed using Spearman's rank correlation test (two-tailed).For paired non-parametric multiple comparisons, Dunn's test with ANOVA was used to analyze differences among nonparametric data for paired samples.ROC curve analysis was utilized to determine AUC and optimal cut-off value for each kit for prediction of endoscopic remission.According to the optimal cut-off value, values for sensitivity, specificity, predictive value, and accuracy were also calculated, along with the 95% CI.Statistical analyses were performed using GraphPad Prism software (version 10.0.3), except for DeLong test and multiple regression analysis results, which were analyzed using the EZR software package (version 1.61, modified version of R commander version 2.8-0).

Table 2 .
Correlation analysis between FC kits in all samples.FC fecal calprotectin, CI confidence interval.

Table 5 .
Sensitivity, specificity, predictive value, and accuracy of FC kits for predicting endoscopic mucosal healing in UC patients.FC fecal calprotectin, UC ulcerative colitis, CI confidence interval, PPV positive predictive value, NPV negative predictive value, CI confidence interval.

Table 6 .
Multiple regression analysis of biomarkers for predicting endoscopic remission in UC patients.UC ulcerative colitis, CI confidence interval, CI confidence interval, WBC white blood cell, Hb hemoglobin, Plt platelet, Alb albumin, CRP C-reactive protein, ESR erythrocyte sedimentation rate.